BIOCHEMICAL EVALUATION OF PHOTOLABILE PRECURSORS OF CHOLINE AND OF CARBAMYLCHOLINE FOR POTENTIAL TIME-RESOLVED CRYSTALLOGRAPHIC STUDIES ON CHOLINESTERASES

Acetylcholinesterase and butyrylcholinesterase both rapidly hydrolyze the neurotransmitter acetylcholine. The unusual three-dimensional stru cture of acetylcholinesterase, in which the active site is located at the bottom of a deep and nan ow gorge. raises cogent questions concern ing traffic of the substrate, acetylcholine, and the products, choline and acetate, to and from the active site. Time-resolved crystallograp hy offers a promising experimental approach to investigate this issue but requires a suitable triggering mechanism to ensure efficient and s ynchronized initiation of the dynamic process being monitored. Here we characterize the properties of two photolabile triggers which may ser ve as tools in time-resolved crystallographic studies of the cholinest erases. These compounds are 2-nitrobenzyl derivatives of choline and o f carbamylcholine, which generate choline and carbamylcholine, respect ively, upon photochemical fragmentation, Both photolabile compounds ar e reversible inhibitors, which bind at the active sites of acetylcholi nesterase and butyrylcholinesterase with inhibition constants in the m icromolar range, and both photofragmentation processes occur rapidly a nd with a high quantum yield, without substantial photochemical damage to the enzymes, Photolysis both of acetylcholinesterase and of butyry lcholinesterase, complexed with a 2-nitrobenzyl derivative of choline, resulted in regeneration of enzymic activity, Photolysis of acetylcho linesterase complexed with the 2-nitrobenzyl derivative of carbamylcho line led to time-dependent inactivation, resulting from carbamylation of acetylcholinesterase, which could be reversed upon dilution, due to decarbamylation. Both sets of experiments demonstrated release of cho line within the active site. In the former case, choline was produced photochemically at the active site, In the latter case, choline was ge nerated enzymatically, within the active site, concomitantly with carb amylation of the acetylcholinesterase. The two photolabile compounds m ay thus serve as complementary probes for time-resolved studies of the route of product release from the active sites of the cholinesterases .